MicroRNA (miRNA) molecules are single stranded and small (~22 nt) RNA molecules that act as masters regulators of the genome. Research on miRNA has increased dramatically since their discovery just over a decade ago and specific miRNA expression patterns are known to influence most cellular activities, including stem cell differentiation and tumor growth. However, the available techniques to detect miRNA are complex and therefore expensive and time consuming, limiting the capacity of researchers and clinicians to validate and use miRNA as disease biomarkers. In addition, their complicated processes normally introduce sequence bias and make them inaccurate and not reproducible. Here, we propose the development of a quantitative detection assay based on Circular- Supercoiling-Biosensors (Patent Pending) for rapid and multiplex detection of miRNA molecules. Circular-Supercoiling-Biosensors (CS-Biosensors) are a novel kind of biosensor based on DNA supercoiling. The new biosensors detect miRNA with single nucleotide specificity without any enzymatic reaction and require only a 20 minute incubation step before electrophoresis detection. In this proposal, we will optimize CS-Biosensors and demonstrate the potential of the biosensors by developing a multiplex miRNA detection panel for tumor analysis and comparing its performance with Taqman RT-qPCR. The first aim focuses on improving the synthesis and application parameters of the biosensors. The second aim focuses on the development and characterization of a method to conduct multiplex miRNA detection. Our overall goal is to develop a powerful and flexible miRNA detection kit essential for miRNA research and clinical diagnostics. The new kit will be better than available tests for medium and low multiplexing applications (1-50 targets). It will have superior quantitative performance and will be faster, simpler to use and less expensive.